Molecular cloning of ozone-inducible protein from Pinus sylvestris L. with high sequence similarity to vertebrate 3-hydroxy-3-methylglutaryl-CoA-synthase.

We have isolated a Pinus sylvestris cDNA encoding a globular protein of 474 amino acids with a predicted molecular weight of 52,995 Da. The deduced amino acid sequence showed 41.9% identity and 13.6% similarity to mammalian cytosolic 3-hydroxy-3-methylglutaryl-CoA-synthase (HMGS). Treatment of Scots pine seedlings with ozone resulted in a transient increase of a 1.95 kb transcript, whereas a 1.2 kb mRNA decreased transiently, indicating a possible influence of ozone on isoprenoid biosynthesis.

Bioavailability and selected pharmacokinetic parameters of clindamycin hydrochloride after administration of a new 600 mg tablet formulation.

The study was conducted to investigate the pharmacokinetics and relative bioavailability of clindamycin after administration of two oral clindamycin HCl formulations. A new tablet preparation containing 600 mg clindamycin (Clinda-saar 600, test) was compared to a marketed capsule containing 300 mg clindamycin (Sobelin 300, reference). Both preparations revealed comparable in vitro dissolution profiles with high batch conformity and homogeneity. Twenty healthy male volunteers received single doses of 600 mg clindamycin (test: 1 tablet, reference: 2 capsules) in an open, randomized, two-period crossover design. Blood samples were drawn up to 14 h p.a. and clindamycin plasma concentrations were measured using a sensitive and specific HPLC-UV method. Pharmacokinetic characteristics were similar for both preparations, arithmetic mean values (standard deviation) were computed as: AUC(0-infinity) 12.2 (4.2) and 13.1 (4.6) microg x h/ml, Cmax 3.1 (0.8) and 3.4 (0.8) microg/ml, t(max) 0.83 (0.24) and 0.85 (0.34) h, t(1/2) 2.3 (0.4) and 2.3 (0.6) h for test and reference, respectively. Mean relative bioavailability (point estimate) was 93% for AUC and 91% for Cmax. 90% confidence intervals for AUC and Cmax were within the predefined bioequivalence acceptance limits. Bioequivalence of test and reference preparations could be demonstrated. Single doses of 600 mg clindamycin orally were well tolerated without relevant differences between both preparations.

Oxaceprol is as effective as diclofenac in the therapy of osteoarthritis of the knee and hip.

In this multicentre (five centres in Germany), randomised, double-blind, comparative study, 150 patients with painful degenerative joint disease according to EULAR criteria received either oxaceprol (200 mg three times daily) or diclofenac (25 mg three times daily) for 20 days. Joint function, the primary variable, assessed according to Lequesne's indices, improved equally in both treatment groups to a clinically relevant degree. Joint mobility improved by approximately 60% in both groups. By the end of therapy in both groups, the period of pain-free walking time had more than doubled and subjectively evaluated pain perception (VAS) was reduced by almost 50% without any significant differences between the treatments. The incidence of adverse drug reactions was similar in both groups but oxaceprol induced milder symptoms. Oxaceprol is as effective and better tolerated than diclofenac in the treatment of osteoarthritis.

Oxaceprol is a well-tolerated therapy for osteoarthritis with efficacy equivalent to diclofenac.

The therapeutic equivalence and safety of treatment for 21 days with 400 mg t.i.d. oxaceprol (n = 132) and 50 mg t.i.d. diclofenac (n = 131) were assessed in a multicentre, randomised, double-blind study of a mixed population of patients with osteoarthritis of the knee and/or hip. In a per-protocol analysis of efficacy, the mean Lequesne index decreased by 2.5 points in the oxaceprol group (n = 109) and by 2.8 points in the diclofenac group (n = 109). The 95% confidence interval for the end-point difference revealed therapeutic equivalence. This was confirmed by assessments (visual analogue scale) of pain at rest, weight-bearing pain, pain on standing and pain on movement, all of which decreased to a similar extent under both treatments. The pain-free walking time increased in both groups from 10 min to 25 min by the end of the treatment period. Mobility was also increased to a similar extent by both drugs. The physicians assessed treatment as good or very good in 45-46% of patients in both groups. In all patients who received treatment, 28 and 37 adverse events were reported by 25 out of 132 (18.9%) and 33 out of 131 (25.2%) patients treated with oxaceprol and diclofenac, respectively. In 15 patients (11.4%) with 15 adverse events in the oxaceprol group and 25 patients (19.1%) with 27 adverse events in the diclofenac group, a relation to the medication was considered probable. The difference between the groups was statistically significant (p = 0.04106) for the number of these adverse events. Oxaceprol is therapeutically equivalent to diclofenac, but better tolerated than diclofenac in the treatment of osteoarthritis.

A new variant of B1 elements, vB1, identified upstream of a retroviral integration site.

A new variant of the murine B1 retroposon family, vB1, was identified in antisense orientation 165 bases upstream of the integration site of RFB MuLV in infected NIH3T3 fibroblasts. vB1 revealed a characteristic B1 structure, but contained two additional unique repeats. This variant facilitates identification of related vB1 elements which are considered to represent a minor subgroup of B1 elements.

Enrichment of chromosome specific hncDNAs by magnetic bead coupled Alu sequences.

We have employed a strategy for the rapid enrichment of cDNA clones from human chromosome 22 utilizing magnetic beads. Starting from a somatic cell hybrid which retains chromosome 22 in rodent background, heteronuclear (hn) RNA was transcribed into hncDNA using poly dT-primers. Using linker specific primers hncDNA was amplified by PCR. To identify chromosome 22 specific hncDNAs a highly human specific Alu consensus sequence (PD39) was biotinylated and hybridized to the PCR product of the hncDNAs in solution. Hybridized hncDNA-PD39 complexes are captured using streptavidin-coated magnetic beads. Hybridized hncDNAs are selectively amplified by PCR. To verify the chromosome specificity the hncDNA was used as probe for in situ hybridization. Following two rounds of selection with magnetic beads there was an increasingly strong hybridization signal on chromosome 22. The capturing of hncDNAs by magnetic beads as described in this study is faster and more efficient than previously described methods for the isolation of chromosome specific hncDNAs. The novel approach has been employed to generate hncDNAs highly enriched for chromosome 22 specific sequences.

Molecular analysis of four lactate dehydrogenase-A mutants in the mouse.

Four electrophoretic and/or enzyme-activity variants of murine LDH-A subunit (Ldhla-m1Neu, Ldhla-m5Neu, Ldhla-m6Neu, Ldhla-m9Neu), induced by procarbazine hydrochloride or ethylnitrosourea (ENU), were analyzed at the DNA level. The exons of the Ldhl gene from homozygous mutants were amplified by PCR and sequenced. Three mutations resulted from nucleotide substitutions in exon 5: the transitions A-->G at codons 216 (Ldhla-m5Neu) and 225 (Ldhla-m6Neu), and the transversion G-->C (Ldhla-m1Neu) at codon 222. The mutations resulted in the replacements of Glu by Gly (Ldhla-m5Neu), Gln by Arg (Ldhla-m6Neu) and Asp by His (Ldhla-m1Neu). The fourth base substitution, the transition T-->C (Ldhla-m9Neu), has been found at the GT donor splice site following the first exon; this mutation affected the efficiency of transcription. All ENU-induced mutations were A/T-->G/C transitions. The mutation events could be correlated with the biochemical and physiological alterations observed in affected mice.

Down-modulation of HIV-1 LTR activity by an extra-LTR nef gene fragment.

The objective of this study was to assess the inhibitory effect of potential negative regulatory elements on human immunodeficiency virus (HIV)-1 long terminal repeat (LTR) activity. This was carried out by pairwise comparisons of reporter gene activities of HIV-LTR-CAT constructs differing in the presence and absence of nef sequences in transient transfection assays. Parallel transfections were performed in two persistently HIV-infected cell lines and the uninfected parental cell lines. The negative regulatory element (NRE) of the LTR did not suppress HIV LTR activity in any of the cell lines examined. However, a non-LTR-derived fragment of the nef gene had a distinct suppressive effect on activity of the full-length LTR in chronically infected astrocytoma cells. A weaker negative effect of this nef partial sequence (nps) was detected in the other cell lines with constructs lacking the NRE. The nps was capable of suppressing LTR activity in trans in chronically infected astrocytoma cells in a concentration-dependent manner. These results stress a negative role of a non-LTR nef partial sequence in a cell-specific manner. In addition, our data indicate that nps functions in trans with promoters unrelated to HIV LTR such as SV40 early and CMV immediate-early promoters.

Cloning, sequencing, mapping and hyperexpression of the ribC gene coding for riboflavin synthase of Escherichia coli.

The gene coding for riboflavin synthase of Escherichia coli has been cloned by marker rescue on a 6-kb fragment that has been sequenced. The riboflavin synthase gene is identical to the ribC locus and codes for a protein of 213 amino acids with a mass of 23.4 kDa. It was mapped to a position at 37.5 min on the physical map of the E. coli chromosome. The 3' end of the ribC gene is directly adjacent to the cfa gene, which codes for cyclopropane-fatty-acid synthase. This gene is followed by two open reading frames designated ydhC and ydhB, which are predicted to code for putative proteins with 403 amino acids and 310 amino acids, respectively. The gene ydhC is similar to genes coding for resistance against various antibiotics (cmlA, bcr) and probably codes for a transmembrane protein. The protein specified by ydhB shows sequence similarity to a large family of DNA-binding proteins and probably represents a helix-turn-helix protein. The ydhB gene is directly adjacent to the regulatory gene purR. A 288-bp segment of the cfa gene has earlier been mapped incorrectly to a position adjacent to greA at 67 min. The ribC gene was hyperexpressed in recombinant E. coli strains to a level of about 30% of cellular protein. The protein was purified to homogeneity by chromatography. The specific activity was 26000 nmol.mg-1.h-1. The protein sediments at a velocity of S20 = 3.8 S. Sedimentation-equilibrium centrifugation indicated a molecular mass of 70 kDa, consistent with a trimer structure. The primary structure of riboflavin synthase is characterized by internal sequence similarity (25 identical amino acids in the C-terminal and N-terminal parts suggesting two structurally similar folding domains.

Two groups of endogenous MMTV related retroviral env transcripts expressed in human tissues.

The human genome contains at least 50 copies of the human endogenous retrovirus K (HERV-K) family which is related to the mouse mammary tumor virus (MMTV). Some members have been shown to be transcriptionally active and to have large open reading frames. Using the RT-PCR method we have investigated the HERV-K env transcription pattern in several malignant tissues and in peripheral blood mononuclear cells PBMCs). Samples were derived from chronic myelogenous leukemia (CML), breast cancer, colon cancer, high and low grade non-Hodgkin's lymphomas, Hodgkin's disease, myelodysplastic syndrome, thyroid adenoma (TA) and from PBMCs of patients with breast cancer, gastric cancer, and of healthy individuals. We found abundant HERV-K env transcripts in all tissues under investigation. Using HERV-K 10 specific primers for amplification we detected in addition to transcripts with high homology to HERV-K 10 (ca. 96% homology on the amino acid level) also transcripts of low homology to HERV-K10 (ca. 71%). Interestingly, all solid tissues containing high percentages of malignant cells such as breast cancer, colon carcinoma, low and high grade non-Hodgkin's lymphomas showed exclusively HERV-K env related transcripts with low homology to HERV-K 10. In contrast, in samples containing only a low proportion of malignant cells or no malignant cells at all we observed both types of transcripts. Thus, our data suggest that the expression pattern of HERV-K elements in human cells is very heterogenous and subjected to a complex transcriptional regulation.

Molecular and pathogenic characterization of the RFB osteoma virus: lack of oncogene and induction of osteoma, osteopetrosis, and lymphoma.

RFB virus is an ecotropic C-type retrovirus isolated from CF-1 mice, in which it is associated with induction of osteomas. Sequence analysis of the RFB provirus revealed no evidence for presence of an oncogene or a recombined env gene. RFB virus is a member of the murine leukemia virus (MuLV) group (RFB MuLV), sharing 97% nucleotide identity with the endogenous ecotropic provirus of AKR mice (Akv). Like Akv, expression of RFB MuLV mRNAs is inducible by dexamethasone treatment, indicating that FRB MuLV also shares transcriptional control signals with Akv. We assessed the pathogenic potential of RFB MuLV in NMRI mice, which, in contrast to CF-1 mice, do not contain endogenous ecotropic retroviruses. RFB MuLV induced osteomas, osteopetrosis, and lymphomas in newborn NMRI mice. Another CF-1 mouse-derived leukemia virus, FBJ MuLV, the helper virus of the FBJ osteosarcoma virus stock, as well as Akv, also induced osteomas, osteopetrosis, and lymphomas in NMRI mice similar to RFB MuLV. These findings indicate that endogenous retroviruses carry a pathogenic potential in hematopoietic tissues and in the skeleton.

Isolation and localization of transcribed sequences on human chromosome 22.

Recently, we reported the generation of a heteronuclear (hn) cDNA library from a human x rodent somatic cell hybrid retaining human chromosome 22. Here, we report the characterization and localization of 12 cDNA derived clones from this library. Human-specific cDNA sequences have been selectively amplified by inter-Alu PCR. To exclude Alu transcripts, only clones with inserts larger than 500 bp were analyzed. Ten of the 12 clones were localized by PCR on chromosome 22, with four clones mapping on additional chromosomes. One PCR-localized clone and two additional clones were mapped on chromosome 22 by in situ hybridization. Transcripts in human cells were identified for seven of the eight clones analyzed by RT-PCR. None of the clones showed significant sequence similarities within the GenBank and EMBL databases, indicating that these clones represent previously unknown genes. This is the first report on the isolation of chromosome 22-specific transcripts from a human x rodent somatic cell hybrid.

Onset and dynamics of osteosclerosis in mice induced by Reilly-Finkel-Biskis (RFB) murine leukemia virus. Increase in bone mass precedes lymphomagenesis.

Newborn NMRI strain mice were infected with Reilly-Finkel-Biskis (RFB) murine leukemia virus (MuLV), a murine leukemia virus that has been shown to induce lymphomas, osteosclerosis, and osteomas in susceptible strains of mice. Bone histomorphometry of the distal femoral metaphyses at 3-month intervals showed osteosclerosis 3 (100%), 6 (100%), and 9 (93%) months after infection. This was represented by significantly augmented cancellous bone mass and accompanied by distinct changes in bone architecture. High numbers of provirus copies were detected at 2-4 weeks in femora, humeri, and calvaria, and viral protein was highly expressed in trabecular and cortical bone cells, particularly in osteocytes. Infected mice showed enhanced bone formation and smaller numbers of osteoclasts relative to sex- and age-matched controls. Osteoclastic differentiation was significantly reduced in cocultures of spleen or bone marrow cells with RFB MuLV-infected osteoclastogenic, osteoblast-like cells. However, RFB MuLV did not impair the activity of mature osteoclasts. In infected mice lymphomas were only observed at 6 (22%) and 9 months (40%) of age. At 3 months, IgG gene and TCR-beta gene rearrangements were not detectable, and new proviruses showed a heterogeneous integration pattern, indicating the absence of lymphoma in early osteosclerotic mice. In contrast, lymphomas, which developed in 8- to 9-month-old infected mice, showed IgG rearrangements indicating development of B-cell lymphomas, together with mono- or oligoclonal expansion of distinct patterns of proviral integrations. These results indicate that RFB MuLV-induced osteosclerosis develops within 3 months after infection and precedes lymphomagenesis. It may therefore be considered an independent skeletal lesion in MuLV-infected mice.